Production scale method of forming microparticles

ABSTRACT

The present invention relates to a method for forming microparticles of a material from microdroplets of a solution, wherein the solution comprises the material dissolved in a solvent. The method includes the steps of directing the microdroplets into a freezing zone, wherein the freezing zone is surrounded by a liquified gas, and wherein the microdroplets freeze. The frozen microdroplets are then mixed with a liquid non-solvent, whereby the solvent is extracted into the non-solvent, thereby forming the microparticles.

RELATED APPLICATIONS

[0001] This application is a Continuation of pending U.S. patentapplication Ser. No. 10/006,991, filed on Dec. 6, 2001, which is aContinuation of pending U.S. patent application Ser. No. 09/587,821,filed on Jun. 6, 2000, which is a Continuation of U.S. patentapplication Ser. No. 09/305,413, filed on May 5, 1999, now U.S. Pat. No.6,153,129, which is a Continuation of U.S. patent application Ser. No.08/443,726, filed on May 18, 1995, now U.S. Pat. No. 5,922,253, theentire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Many illnesses or conditions require a constant level ofmedicaments or agents in vivo to provide the most effectiveprophylactic, therapeutic or diagnostic results. In the past,medicaments were given in doses at intervals which resulted influctuating medication levels.

[0003] Attempts to control and steady medication levels have morerecently included the use of many biodegradable substances, such aspolymeric and protein microspheres containing the medicament. The use ofthese microspheres provided an improvement in the controlled release ofmedicaments by utilizing the inherent biodegradability of the polymer toimprove the release of the medicament and provide a more even,controlled level of medication.

[0004] However, many of these methods result in low yields ofmicrospheres due to a combination of the methods and apparatus used.Further, some processes cannot be scaled-up from experimental level to acommercial production level.

[0005] Therefore, a need exists for a method of forming microsphereswith lower losses of biologically active agent, high product yields, andcommercial-scale feasibility.

SUMMARY OF THE INVENTION

[0006] This invention relates to a method for forming microparticles ofa material from microdroplets of a solution, wherein the solutioncomprises the material dissolved in a solvent. The method includes thesteps of directing the microdroplets into a freezing zone, wherein thefreezing zone is surrounded by a liquified gas, and wherein themicrodroplets freeze. The frozen microdroplets are then mixed with aliquid non-solvent, whereby the solvent is then extracted into thenon-solvent, thereby forming the microparticles.

[0007] This invention has numerous advantages, for instance, this methodand apparatus provides high yields, commercial production levels ofcontrolled release microparticles, an enclosed system for asepticprocessing, microparticle size control and process controlreproducibility

[0008] In addition, the method of invention permits greater tailoring oftemperature profiles during performance of the method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 is a cut-away side elevational illustration of an apparatusof the invention suitable for forming microparticles of a material,according to the method of the invention by freezing microdroplets of asolution of the material in a solvent, within a freezing zone cooled byan encircling flow of liquified gas and then extracting the solvent fromthe frozen microdroplets, by exposure to a liquid non-solvent.

[0010]FIG. 2 is a cut-away side elevational illustration of anotherembodiment of an apparatus of the invention suitable for formingmicroparticles of a material according to the method of the invention,by freezing microdroplets of a solution of the material in a solvent,within a freezing zone cooled by an encircling flow of a liquified gasand then extracting the solvent from the frozen microdroplets, byexposure to a liquid non-solvent.

[0011]FIG. 3 is a cut-away side elevational illustration of yet anotherembodiment of an apparatus of the invention suitable for formingmicroparticles of a material according to the method of the invention,by freezing microdroplets of a solution of the material in a solvent,within a freezing zone cooled by an encircling flow of a liquified gasand then extracting the solvent from the frozen microdroplets, byexposure to a liquid non-solvent.

[0012]FIG. 4 is a cut-away side elevational illustration of an alternateembodiment of an apparatus of the invention suitable for formingmicroparticles of a material according to the method of the invention,by freezing microdroplets of a solution of the material in a solvent,within a freezing zone cooled by an encircling flow of a liquified gasand then extracting the solvent from the frozen microdroplets, byexposure to a liquid non-solvent.

[0013] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to a method and apparatus forforming microparticles of a material from a solution of the material. Amicroparticle, as defined herein, comprises a particle of a materialhaving a diameter of less than about one millimeter. A microparticle canhave a spherical, non-spherical or irregular shape. It is preferred thata microparticle be a microsphere.

[0015] Materials suitable to form microparticles of this inventioninclude, for example, polymers, peptides, polypeptides, proteins, smallmolecule drugs and pro-drugs.

[0016] A microparticle can also contain one or more additionalsubstance, which is dispersed within the microparticle. Wherein thematerial comprises a polymer, the polymer solution contains at least onebiologically active agent.

[0017] A biologically active agent, as defined herein, is an agent, or ametabolite of the agent, which possesses therapeutic, prophylactic ordiagnostic properties in vivo, in the form of said agent whenadministered, or after metabolism (e.g., a pro-drug, such ashydrocortisone succinate).

[0018] One embodiment of an apparatus of the invention, suitable forperforming the method of invention, is illustrated in FIG. 1. Saidapparatus includes vessel 10, typically in a cylindrical shape, havingside wall 12, vessel top 14, vessel bottom 16 and internal wall 18. Sidewall 12 and vessel bottom 16 are usually insulated, using conventionalinsulation methods, to minimize heat leakage from the outsideenvironment into vessel 10, thereby providing improved temperaturecontrol within vessel 10. Conventional insulation methods include, forexample, application of at least one layer of insulation material 17 tocover the outside surfaces of side wall 12 and vessel bottom 16. Othermeans of insulating include, for instance, vacuum jacketing side wall 12and vessel bottom 16 with radiation shielding. Suitable insulationmaterials include conventional insulation materials, such as mineralfiber, polystyrene, polyurethane, rubber foams, balsa wood or corkboard.

[0019] In this embodiment, vessel top 14 is typically not insulated,thereby allowing components of said apparatus, disposed at or nearvessel top 14, to be warmed by heat leakage into the vessel 10.Alternately, vessel top 14 may also be insulated with a suitableinsulation material.

[0020] Vessel 10 is fabricated with a material which can withstandconditions during steam sanitizing, of the inside of vessel 10, and canalso withstand the temperatures and gas pressures experienced in vessel10 while performing the method of invention for forming microparticles11. Suitable materials for vessel 10 include, for example, stainlesssteel, polypropylene and glass.

[0021] Vessel 10, in this embodiment, is a single unitary vessel,divided into freezing section 20 and extraction section 22. Freezingsection 20 is disposed within, and substantially enclosed by, side wall12, vessel top 14 and internal wall 18. Extraction section 22 isdisposed within, and substantially enclosed by, side wall 12, vesselbottom 16 and internal wall 18.

[0022] In an alternate embodiment, freezing section 20 and extractionsection 22 comprises separate vessels, wherein the freezing sectionvessel is disposed generally above the extraction section vessel, andwherein the bottom of the freezing section vessel is connected to thetop or to a side of the extraction section vessel.

[0023] Vessel 10 also includes means for directing liquified gas intofreezing section 20 to form liquified gas flow 24. Liquified gas flow 24consists of a spray of liquified gas and/or at least one stream ofliquified gas. Liquified gas flow 24 begins in freezing section 20 at ornear vessel top 14, and then runs in a generally downward direction,toward internal wall 18. Within freezing section 20, at least a portionof liquified gas flow 24 runs substantially parallel with side wall 12.Liquified gas flow 24 is typically disposed at or near side wall 12. Itis preferred that side wall 12 is generally wetted by liquified gas flow24. Furthermore, liquified gas flow 24 substantially encircles freezingzone 26, which is approximately disposed about the radial centerline offreezing section 20. The extent to which liquified gas flow 24 has gapsin the encircling flow about freezing zone 26, is dependent upon thetype and number of liquified gas directing means employed.

[0024] At least one suitable liquified gas directing means is disposedat or near vessel top 14, at a location which is radially displaced fromthe center of vessel top 14. The radial displacement of a liquified gasdirecting means, is sufficient if the liquified gas directing means doesnot significantly interfere with the formation of microdroplets 28, suchas by freezing a portion of a solution from which microdroplets 28 areformed at microdroplet forming means 30, thereby at least partiallyclogging microdroplet forming means 30. A liquified gas directing meanscan also interfere if a significant portion of microdroplets 28 impactsaid liquified gas directing means.

[0025] In the embodiment illustrated in FIG. 1, suitable liquified gasdirecting means include at least two spray nozzles 32 having a linedischarge or preferably a fan discharge (e.g., flood jet atomizer model1/8-K-SS-1, operated with a liquid gas pressure of about 20 psig; SpraySystems Co., Wheaton, Ill.), which are capable of spraying a liquifiedgas to form at least a portion of liquified gas flow 24. Spray nozzles32 are disposed in freezing section 20 at vessel top 14, and are aboutequidistantly spaced at positions approximately located on a circlecentered around the center of vessel top 14, or centered aroundmicrodroplet forming means 30 if radially displaced from said vessel topcenter. The number of spray nozzles 32 used will depend upon the arc ofthe nozzle discharge and the distance from nozzle 32 to the impact pointon side wall 12 of liquified gas flow 24.

[0026] With two spray nozzles 32 equidistantly displaced from the centerof the top of freezing section 20, encircling liquified gas flow 24 willtypically have two gaps about 180° apart, due to the usual inability ofspray nozzle 32 to spray in greater than a 180° arc. In a preferredembodiment, at least three spray nozzles are disposed in freezingsection 20 to form liquified gas flow 24 which encircles freezing zone26 typically without any significant gaps in the encircling flow.

[0027] Typically, three spray nozzles 32, equidistantly spaced willprovide a 360° liquified gas flow 24. In a more preferred embodiment,six spray nozzles are equidistantly disposed about the center offreezing section 20.

[0028] A liquified gas directing means receives liquified gas, from atleast one liquified gas inlet 34. Liquified gas inlet 34 provides fluidcommunication between liquified gas source 36 and the liquified gasdirecting means. It is understood that other suitable liquified gasintroduction means, capable of directing liquified gas flow into theliquified gas directing means, can be used in place of, or incombination with, liquified gas inlet 34.

[0029]FIG. 2 illustrates another embodiment of a suitable liquified gasdirecting means of an apparatus of this invention. The apparatus of FIG.2 has many of the same elements of FIG. 1 and like elements aredesignated with like numerals. In said apparatus, suitable liquified gasdirecting means comprises weir 102 and liquified gas space 104. Weir 102is disposed within freezing section 20, between side wall 12 andfreezing zone 26. Weir 102 extends from internal wall 18, or alternatelyfrom side wall 12, and extends upwards toward vessel top 14. In oneembodiment, the top portion of weir 102 does not contact vessel top 14,thus permitting liquified gas to flow over the top of weir 102 andfurther into freezing section 20. Alternately, wherein weir 102 contactsvessel top 14, weir 102 is porous or slotted at the top of weir 102 (notshown) to permit liquified gas to flow through the top section of weir102, and further into freezing section 20.

[0030] Liquified gas space 104 is disposed within freezing section 20,between weir 102 and side wall 12. Liquified gas space 104 receivesliquified gas from at least one liquified gas inlet 34. The liquifiedgas is then directed over or through weir 102 further towards the centerof freezing section 20.

[0031] Referring back to FIG. 1, vessel 10 also includes microdropletforming means 30, disposed in freezing section 20 at vessel top 14, forforming microdroplets 28 from a suitable solution. A microdroplet isdefined herein as a drop of solution which, after freezing andsubsequent extraction of the solution's solvent, will form amicroparticle. Examples of suitable microdroplet forming means 30include atomizers, nozzles and various gauge needles. Suitable atomizersinclude, for example, external air (or gas) atomizers (e.g., ModelSUE15A; Spray Systems Co., Wheaton, Ill.), internal air atomizers (e.g.,SU12; Spray Systems Co.), rotary atomizers (e.g., discs, bowls, cups andwheels; Niro, Inc., Columbia, Md.), and ultrasonic atomizers (e.g.,Atomizing Probe 630-0434; Sonics & Materials, Inc., Danbury, Conn.).Suitable nozzles include pressure atomization nozzles (e.g, Type SSTCWhirl Jet Spray Drying Nozzles; Spray Systems Co., Wheaton, Ill.).Typical gauges of needles used to form microdroplets 28 include needleswith gauges between about 16 and about 30.

[0032] In a preferred embodiment, microdroplet forming means 30 is anair atomizer, which can form microparticles 11 having a range ofdiameters between about 1 micrometer, or less, and about 300micrometers. Average microparticle size can be changed by adjusting thepressure of the atomizing gas, supplied to an air atomizer (e.g.,nitrogen gas). Increased gas pressure results in smaller averagemicroparticle diameters.

[0033] Microdroplet forming means 30 is fabricated from a material, orcombination of materials, which can withstand steam sanitizing and alsothe cold temperatures experienced in freezing section 20.

[0034] Microdroplet forming means 30 receives solution from at least onesolution inlet 38. Solution inlet 38 provides fluid communicationbetween solution source 40 and freezing section 20. It is understoodthat other suitable solution introduction means, such as a lance or another device capable of injecting a solution into a cold environment,can be used in place of, or in combination with, solution inlet 38.

[0035] Vessel 10 also includes at least one three-phase port 42, whichis disposed at internal wall 18, and provides fluid communicationbetween freezing section 20 and extraction section 22. Three-phase port42 is sized to allow the flow of a combination of frozen microdroplets44, liquified gas and volatilized gas from freezing section 20 intoextraction section 22.

[0036] Extraction section 22 includes means for separating a liquifiedgas from frozen microdroplets 44. In one embodiment, a suitableseparating means comprises a means for heating extraction section 22,which then volatilizes the liquified gas, thus separating it from frozenmicrodroplets 44, usually contained within the lower portion ofextraction section 22. Said heating means can also be used to warm thesolvent frozen within frozen microdroplets 44. Suitable means forheating can include heat leakage from the outside environment, throughside wall 12 and vessel bottom 16. Optionally, heating means caninclude, for example, electrical means such as heating coils, orrecirculating heat exchanger tubes 46, through which a fluid can becirculated to control temperature within extraction section 22 to firstvolatilize the liquified gas, and then subsequently warm the solvent infrozen microdroplets 44 to control solvent extraction rate.

[0037] An alternate separating means comprises filtered bottom tap 48,which extends from the lower portion of extraction section 22. Filteredbottom tap 48, which contains filter 50, having a pore size less thanthe diameter of microparticles 11, typically ≦1 micrometer, is suitablefor removing liquids, such as liquified gas, from extraction section 22,while retaining frozen microdroplets 44, and possibly microparticles 11,within extraction section 22.

[0038] Gas outlet 52, which is disposed in extraction section 22 atinternal wall 18, is suitable for directing gas, produced byvolatilizing liquified gas, out of vessel 10. Gas outlet 52 canoptionally include a means for reducing pressure within vessel 10, forexample a vacuum blower (e.g., CP-21 low temperature blower, BarberNichols, Arvada, Colo.) or vacuum pump (e.g. E2M18 vacuum pump, EdwardsHigh Vacuum International, Crawley, West Sussex, England) suitable forimpelling gases. Furthermore, gas outlet 52 typically includes filter 53(e.g., a 0.2 micrometer sterile filter) in the gas flow path to supportan aseptic process and provide assurance that formed microparticles 11meet sterility requirements.

[0039] Vessel 10 can optionally include gas outlets 52, disposed inextraction section 22 and/or freezing section 20 (not shown). It ispreferred that no gas outlets be disposed in freezing section 20, as gasventing out of freezing section 20 can result in gas circulationcurrents which can reduce the yield of microparticles 11 produced.

[0040] In addition, vessel 10 can optionally include at least oneoverpressure protection device (not shown), to protect the materialintegrity of vessel 10 from overpressurization caused by thevolatilization of a liquified gas. Typical overpressure protectiondevices include, for instance, rupture disks or pressure relief valves.

[0041] Extraction section 22 also includes at least one non-solventinlet 54, disposed at internal wall 18 and/or in side wall 12.Extraction section 22 receives a liquid non-solvent from non-solventinlet 54 in a stream or spray. Preferably, non-solvent in extractionsection 22 forms extraction bath 56, which is disposed in at least thelower portion of extraction section 22. Non-solvent inlet 54 providesfluid communication between cold non-solvent source 58 and extractionbath 56. It is understood that other suitable means, for introducing aliquid into a vessel under cold conditions, such as a lance or an otherdevice capable of introducing a liquid under cold conditions, can beused in place of, or in combination with, non-solvent inlet 54.

[0042] In another embodiment, a suitable mixing means 60 for mixingfrozen microdroplets 44 and non-solvent, is disposed in extraction bath56. Mixing means 60 is provided to reduce the potential for formation ofextraction gradients within extraction bath 56, such as could occur iffrozen microdroplets 44 clumped at the bottom of extraction section 22.Examples of suitable mixing means 60 include low shear mixing devices,such as a turbine (e.g., Lightning Sealmaster P6X05E with an A310impeller operating at about 0-175 rpm), a marine impeller, a paddlemixer or an external recirculation loop having a low shear pump.

[0043] Vessel 10 further includes bottom tap 62, which extends from thelower portion of extraction section 22. Bottom tap 62 is suitable forremoving microparticles 11 and liquids, such as non-solvent, from vessel10. Alternatively, dip tubes (not shown) may be used to removemicroparticles 11 and liquids from vessel 10.

[0044] When required for drug delivery, relevant internal portions ofthe apparatus of this invention are cleaned and sanitized, orsterilized, between each use to assure the sterility of the finalproduct.

[0045] In the method of this invention, microparticles of a material areformed from a solution of the material in a suitable solvent. Materialssuitable for use in this method can include any soluble materials,provided a non-solvent is available which has a lower melting point thanthe solvent, and which has sufficient miscibility with the solvent toextract solid and/or thawed liquid solvent from a frozen microparticle.Preferably, materials used in this method include peptides,polypeptides, proteins, polymers, small molecule drugs and pro-drugs.

[0046] Any type of suitable polymer can also be used to form amicroparticle. In a preferred embodiment, a polymer used in this methodis biocompatible. A polymer is biocompatible if the polymer, and anydegradation products of the polymer, such as metabolic products, arenon-toxic to humans or animals, to whom the polymer was administered,and also present no significant deleterious or untoward effects on therecipient's body, such as an immunological reaction at the injectionsite. Biocompatible polymers can be biodegradable polymers,non-biodegradable polymers, a blend thereof or copolymers thereof.

[0047] Suitable biocompatible, non-biodegradable polymers include, forinstance, polyacrylates, polymers of ethylene-vinyl acetates and otheracyl substituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonate polyolefins, polyethylene oxide, blends andcopolymers thereof.

[0048] Suitable biocompatible, biodegradable polymers include, forexample, poly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s,poly(lactic acid)s, poly(glycolic acid)s, polycarbonates,polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters,polyacetals, polycyanoacrylates, polyetheresters, polycaprolactone,poly(dioxanone)s, poly(alkylene alkylate)s, polyurethanes, blends andcopolymers thereof. Polymers comprising poly(lactides), copolymers oflactides and glycolides, blends thereof, or mixtures thereof are morepreferred. Said polymers can be formed from monomers of a singleisomeric type or a mixture of isomers.

[0049] A polymer used in this method can be blocked, unblocked or ablend of blocked and unblocked polymers. An unblocked polymer is asclassically defined in the art, specifically having free carboxyl endgroups. A blocked polymer is also as classically defined in the art,specifically having blocked carboxyl end groups. Generally, the blockinggroup is derived from the initiator of the polymerization reaction andis typically an alkyl radical.

[0050] Acceptable molecular weights for polymers used in this inventioncan be determined by a person of ordinary skill in the art taking intoconsideration factors such as the use of the microparticle, the desiredpolymer degradation rate, physical properties such as mechanicalstrength, and rate of dissolution of polymer in solvent. Typically, anacceptable range of molecular weights for polymeric microparticleshaving therapeutic uses is between about 2,000 Daltons to about2,000,000 Daltons.

[0051] In an even more preferred embodiment, the polymer is apoly(lactide-co-glycolide) with a lactide:glycolide ratio of about 1:1and a molecular weight of about 5,000 Daltons to about 70,000 Daltons.In an even more preferred embodiment, the molecular weight of thepoly(lactide-co-glycolide) used in the present invention has a molecularweight of about 5,000 Daltons to about 42,000 Daltons.

[0052] Typically, a suitable polymer solution contains between about 1%(w/w) and about 30% (w/w) of a suitable biocompatible polymer, whereinthe biocompatible polymer is typically dissolved in a suitable polymersolvent. Preferably, a polymer solution contains about 5% (w/w) to about20% (w/w) polymer.

[0053] Microparticles can be formed by either a continuous freezing andextraction process or by a batch process wherein a batch of frozenmicrodroplets is formed in a first step, and then in a separate secondstep, the frozen microdroplets in the batch are extracted to formmicroparticles.

[0054] In this method, freezing zone 26 includes the portion of freezingsection 20, which is substantially encircled by liquified gas flow 24.Freezing zone 26 is formed within freezing section 20 of vessel 10, bydirecting a flow 24 of a suitable liquified gas from at least two spraynozzles 32 in a substantially downward direction, toward side wall 12.Typically, the liquified gas discharged from spray nozzles 32 is angledsuch that the liquified gas impinges against side wall 12 to formliquified gas flow 24 along the inside surface of side wall 12, thuswetting side wall 12. In a preferred embodiment, liquified gas, fromeach of six spray nozzles 32, is directed against side wall 12 at anangle to side wall 12 of less than about 30° to reduce the splashing ordeflection of liquified gas off of side wall 12.

[0055] Alternately, liquified gas flow 24 is directed substantiallyparallel to, but displaced from the inside surface of side wall 12 toeffectively form an independent wall of liquified gas extending fromspray nozzles 32 to inner wall 18.

[0056] Liquified gas is provided to spray nozzles 32 from liquified gassource 36 through liquified gas inlet 34.

[0057] Liquified gases suitable for use in this method include liquidargon (−85.6° C.), liquid nitrogen (−95.8° C.), liquid helium or anyother liquified gas having a temperature sufficiently low to freezemicrodroplets 28 of a solution, while the microdroplets 28 are containedin freezing zone 26 or in liquified gas flow 24. Liquid nitrogen ispreferred.

[0058] In an alternate embodiment, illustrated in FIG. 2, freezing zone24 is formed within freezing section 20, by directing liquified gas fromliquified gas source 36, through liquified gas inlet 34 and intoliquified gas space 104, wherein the liquified gas then flows up overweir 102, or through slots (not shown) in weir 102 to form liquified gasflow 24. Liquified gas flow 24 then flows substantially downward alongthe inside surface of weir 102.

[0059] Referring back to FIG. 1, microdroplets 28 of a solution,preferably a solution of a polymer, are then directed through freezingzone 26, in a substantially downward direction, wherein microdroplets 28freeze to form frozen microdroplets 44. A portion of microdroplets 28may freeze by contacting liquified gas in liquified gas flow 24.Microdroplets 28 were previously formed by directing the solution fromsolution source 40, through solution inlet 38, into a suitablemicrodroplet forming means 30. Typically, within freezing section 20, atleast a portion of the liquified gas will volatilize due to heatin-leakage and/or heat transfer from microdroplets 28 to the liquifiedgas.

[0060] A three-phase flow of volatilized gas, liquified gas and frozenmicrodroplets 44 then flows from the bottom of freezing section 20 andinto extraction section 22, through three-phase port 42.

[0061] In one embodiment, at least a portion of frozen microdroplets 44are entrained within liquified gas flow 24, which then carry frozenmicrodroplets 44 into extraction section 22. The entrainment of frozenmicrodroplets 44 within liquified gas flow 24 may improve the finalyield of microparticles 11 produced, according to the method ofinvention, by transporting, into extraction section 22, frozenmicrodroplets 44 which might otherwise remain within freezing section20, such as by adhering to side wall 12 and/or internal wall 18, and/orby reducing the loss of airborne frozen microdroplets 44 from vessel 10through gas outlet 52.

[0062] The liquified gas is then separated from frozen microdroplets 44by suitable separating means, leaving frozen microdroplets 44 disposedin the lower portion of extraction section 22.

[0063] In one embodiment, the liquified gas is heated to a temperaturebelow the melting point of frozen microdroplets 44, but at or above theboiling point of the liquified gas whereby the liquified gas isvaporized and separated from frozen microdroplets 44.

[0064] Alternately, liquified gas can be separated by pulling a partialvacuum on extraction section 22 through gas outlet 52 and heating theliquified gas to a temperature below the boiling point of the liquifiedgas but high enough to elevate the vapor pressure of the liquified gas,thereby evaporating the liquified gas.

[0065] After heating, the liquified gas is volatilized, therebyseparating the liquified gas from frozen microdroplets 44. The liquifiedgas can be heated by heat in-leakage from the outside environmentthrough side wall 12 and vessel bottom 16. Preferably, extractionsection 22 is heated by an electrical heat source or by recirculating awarmer fluid, such as nitrogen gas or a nitrogen gas/liquified nitrogenmixture, through heat exchanger tubes 46. In addition, a fluid can becirculated, through heat exchanger tubes 46, to control temperaturewithin extraction section 22 to firstly volatilize the liquified gas ina controlled manner, and then subsequently to slowly warm the solvent infrozen microdroplets 44 to permit solvent extraction into the liquidnon-solvent.

[0066] Alternately, liquified gas is separated from frozen microdroplets44 by directing the liquified gas through filter 50 and then out ofextraction section 22 through filtered bottom tap 48. Directing theliquified gas through filter 50 thereby removes the liquified gas fromextraction section 22, while retaining frozen microdroplets 44 withinthe bottom portion of extraction section 22.

[0067] Wherein the liquified gas is separated by heating to volatilizethe liquified gas, the resulting volatilized gas is then directed out ofextraction section 22 through at least one gas outlet 52. Pressurewithin vessel 10 is primarily dependent upon the amount of liquifiedgas, which is volatilized within extraction section 22, and upon thedischarge rate of gas through gas outlet 52. Vessel 10 can be operatedat pressures above, equal to, or below atmospheric pressure. The upperpressure limit for performing this method is dependent upon the pressurerating of vessel 10.

[0068] It is preferred that the method of invention be performed, duringformation of frozen microdroplets 44, under a partial vacuum. Achievinga partial vacuum within extraction section 22, and thus throughoutvessel 10, is achieved by means known to one of skill in the art, suchas using a pump or blower to take a suction through gas outlet 52 onextraction section 22.

[0069] Following separation of frozen microdroplets 44 from theliquified gas, frozen microdroplets 44 are then contacted with asuitable cold liquid non-solvent, which is at a temperature below themelting point of frozen microdroplets 44. In a preferred embodiment, theliquid non-solvent is maintained below the melting point of frozenmicrodroplets 44, and the solvent is extracted from the solid state intothe liquid non-solvent to form porous microparticles 11 over a period ofabout 1 to about 24 hours. The extraction of solid state solvent slowsthe extraction process, thereby providing greater control of extractionand microparticle 11 formation.

[0070] In another embodiment, the liquid non-solvent is warmed to atemperature at or above the melting point of frozen microdroplets 44.The solvent in frozen microdroplets 44 thereby thaws and then, isextracted into the non-solvent. The solvent is thereby extracted as asolid and/or a liquid depending upon the various factors such as thevolume of solvent in frozen microdroplet 44, the volume of non-solventto which frozen microdroplet 44 is exposed, and the warming rate offrozen microdroplet 44. Depending upon the warming rate, themicroparticle produced can also be porous, for lower warming rates, orsignificantly less porous due to partial particle condensation followingrapid solvent extraction.

[0071] Non-solvent can be in the form of a spray, a stream and/orextraction bath 56. Preferably, frozen microdroplets 44 are immersedwithin the non-solvent of extraction bath 56.

[0072] Suitable non-solvents are defined as non-solvents of the materialin solution, which are sufficiently miscible with the solvent of thesolution to extract said solvent, out of the frozen microdroplets 44 asthe solvent warms, thereby forming microparticles 11. In addition, thenon-solvent has a melting point below the melting point of the frozenmicrodroplets 44.

[0073] In another embodiment, second non-solvents, such as hexane, areadded to the first non-solvent, such as ethanol, to increase the rate ofsolvent extraction from certain polymers, such aspoly(lactide-co-glycolide).

[0074] In a preferred embodiment, at least a portion of frozenmicrodroplets 44 are entrained within the non-solvent, which may improvethe final yield of microparticles 11 produced, according to the methodof invention, by transporting frozen microdroplets 44 into extractionbath 56. The frozen microdroplets may otherwise have been lost in theprocess due to adhering to side wall 12, and/or from the loss ofairborne frozen microdroplets 44 from vessel 10 through gas outlet 52.

[0075] In a further embodiment, frozen microdroplets 44 are thenagitated within extraction bath 56 by mixing means 60 to reduce theconcentration gradient of solvent within the non-solvent surroundingeach frozen microdroplet 44 or microparticle 11, thereby improving theeffectiveness of the extraction process.

[0076] In yet another embodiment, the extraction process includes thesequential addition to, and drainage of separate aliquots of non-solventfrom, extraction section 22, to extract solvent into each separatealiquot. Extraction is thereby performed in a step-wise manner. Thawingrate is dependent on the choice of solvents and non-solvents, and thetemperature of the non-solvent in extraction section 22. Table 1provides exemplary polymer/solvent/non-solvent systems that can be usedin this method along with their melting points. TABLE 1 AppropriatePolymer Solvents and Non-Solvents Systems, with Solvent and non-SolventMelting Points POLYMER SOLVENT (° C.) NON-SOLVENT (° C.) Poly(lactide)Methylene Ethanol (−114.5) Chloride (−95.1) Chloroform (−63.50) Methanol(−97.5) Poly(lactide-co- Ethyl Ethanol (−114.5) glyco-lide) Acetate(−83.6) Acetone (−95.4) Ethyl ether (−116.3) Methylene Pentane (−130)Chloride (−95.1) Isopentane (−160) Poly(capro- Methylene Ethanol(−114.5) lactone) Chloride (−95.1) Poly (vinyl Water (0) Acetone (−95.4)alcohol) Ethylene-vinyl Methylene Ethanol (−114.5) acetate Chloride(−95.1)

[0077] For proteins it is preferred that frozen microdroplets 44 beslowly thawed while the polymer solvent is extracted to produce amicroparticle.

[0078] A wide range of sizes of microspheres can be made by varying thedroplet size, for example, by changing the nozzle diameter or air flowinto an air atomizer. If very large diameters of microparticles 11 aredesired, they can be extruded through a syringe directly into freezingzone 24. Increasing the inherent viscosity of the polymer solution canalso result in an increasing microparticle size. The size ofmicroparticles 11 produced by this process can range from greater thanabout 1000 down to about 1 micrometer, or less, in diameter. Usually, amicroparticle will be of a size suitable for injection into a human orother animal. Preferably, the diameter of a microparticles 11 will beless than about 180 micrometers.

[0079] Following extraction, microparticles 11 are filtered and dried toremove non-solvent, by means known to one of skill in the art. For apolymeric microparticle, said microparticle is preferably not heatedabove its glass transition temperature to minimize adhesion betweenmicroparticles, unless additives, such as mannitol, are present toreduce adhesion between the microparticles.

[0080] In another embodiment, a solution of a material also contains oneor more additional substance, which is dispersed within the solution.Said additional substance is dispersed by being co-dissolved in thesolution, suspended as solid particles, such as lyophilized particles,within the solution, or dissolved in a second solvent, which isimmiscible with the solution, and is mixed with the solution to form anemulsion. Solid particles, suspended in the solution can be largeparticles, with a diameter greater than 300 micrometers, or micronizedparticles with a diameter as small as about 1 micrometer. Typically, theadditional substance should not be soluble in the non-solvent.

[0081] Wherein the material comprises a polymer, the polymer solutioncontains at least one biologically active agent. Examples of suitabletherapeutic and/or prophylactic biologically active agents includeproteins, such as immunoglobulin-like proteins; antibodies; cytokines(e.g., lymphokines, monokines and chemokines); interleukins;interferons; erythopoietin; hormones (e.g., growth hormone andadrenocorticotropic hormone); growth factors; nucleases; tumor necrosisfactor; colony-stimulating factors; insulin; enzymes; antigens (e.g.,bacterial and viral antigens); and tumor suppressor genes. Otherexamples of suitable therapeutic and/or prophylactic biologically activeagents include nucleic acids, such as antisense molecules; and smallmolecules, such as antibiotics, steroids, decongestants, neuroactiveagents, anesthetics, sedatives, cardiovascular agents, anti-tumoragents, antineoplastics, antihistamines, hormones (e.g., thyroxine) andvitamins.

[0082] Examples of suitable diagnostic and/or therapeutic biologicallyactive agents include radioactive isotopes and radiopaque agents.

[0083] The microspheres made by this process can be either homogeneousor heterogeneous mixtures of the polymer and the active agent.Homogeneous mixtures are produced when the active agent and the polymerare both soluble in the solvent, as in the case of certain hydrophobicdrugs such as steroids. Heterogeneous two phase systems having discretezones of polymer and active agent are produced when the active agent isnot soluble in the polymer/solvent, and is introduced as a suspension oremulsion in the polymer/solvent solution, as with hydrophilic materialssuch as proteins in methylene chloride.

[0084] The amount of a biologically active agent, which is contained ina specific batch of microparticles is a therapeutically,prophylactically or diagnostically effective amount, which can bedetermined by a person of ordinary skill in the art taking intoconsideration factors such as body weight, condition to be treated, typeof polymer used, and release rate from the microparticle.

[0085] In one embodiment, a controlled release polymeric microparticlecontains from about 0.01% (w/w) to approximately 50% (w/w) biologicallyactive agent. The amount of the agent used will vary depending upon thedesired effect of the agent, the planned release levels, and the timespan over which the agent will be released. A preferred range of loadingfor biologically active agents is between about 0.1% (w/w) to about 30%(w/w).

[0086] When desired, other materials can be incorporated intomicroparticles with the biologically active agents. Examples of thesematerials are salts, metals, sugars, surface active agents. Additives,such as surface active agents, may also be added to the non-solventduring extraction of the solvent to reduce the possibility ofaggregation of the microparticles.

[0087] The biologically active agent can also be mixed with otherexcipients, such as stabilizers, solubility agents and bulking agents.Stabilizers are added to maintain the potency of the agent over theduration of the agent's release. Suitable stabilizers include, forexample, carbohydrates, amino acids, fatty acids and surfactants and areknown to those skilled in the art. The amount of stabilizer used isbased on ratio to the agent on a weight basis. For amino acids, fattyacids and carbohydrates, such as sucrose, lactose, mannitol, dextran andheparin, the molar ratio of carbohydrate to agent is typically betweenabout 1:10 and about 20:1. For surfactants, such as the surfactantsTween™ and Pluronic™, the molar ratio of surfactant to agent istypically between about 1:1000 and about 1:20.

[0088] In another embodiment, a biologically active agent can belyophilized with a metal cation component, to stabilize the agent andcontrol the release rate of the biologically active agent from amicroparticle, as described, in co-pending U.S. patent application Ser.No. 08/279,784, filed Jul. 25, 1994, the teachings of which areincorporated herein by reference.

[0089] Solubility agents are added to modify the solubility of theagent. Suitable solubility agents include complexing agents, such asalbumin and protamine, which can be used to control the release rate ofthe agent from a polymeric or protein matrix. The weight ratio ofsolubility agent to biologically active agent is generally between about1:99 and about 20:1.

[0090] Bulking agents typically comprise inert materials. Suitablebulking agents are known to those skilled in the art.

[0091] Further, a polymeric matrix can contain a dispersed metal cationcomponent, to modulate the release of a biologically active agent fromthe polymeric matrix is described, in co-pending U.S. Pat. No.5,656,297, filed May 3, 1994, to Bernstein et al. and in a co-pendingInternational Application designating the United States, PCT/US95/05511,filed May 3, 1995, the teachings of which are incorporated herein byreference.

[0092] In yet another embodiment, at least one pore forming agent, suchas a water soluble salt, sugar or amino acid, is included in themicroparticle to modify the microstructure of the microparticle. Theproportion of pore forming agent added to the polymer solution isbetween about 1% (w/w) to about 30% (w/w). It is preferred that at leastone pore forming agent be included in a non-biodegradable polymericmatrix of the present invention.

[0093]FIG. 3 illustrates yet another embodiment of an apparatus of theinvention, suitable for performing the method of invention. Theapparatus of FIG. 3 has many of the same elements of FIG. 1 and likeelements are designated with like numerals. In this apparatus, freezingsection 20 is disposed within freezing vessel 202, and is substantiallyenclosed by side wall 12, vessel top 14 and freezing vessel bottom 204.Extraction section 22 is disposed, likewise, within extraction vessel206, and is substantially enclosed by, side wall 12 a, extraction vesseltop 208 and vessel bottom 16. Freezing vessel 202 is disposed generallyabove extraction vessel 206. Conduit 210 is disposed between freezingvessel 202 and extraction vessel 206. Conduit 210 includes conduit inlet212, disposed at or near freezing vessel bottom 204, and conduit outlet214, disposed at or near extraction vessel top 208. Conduit 210 providesthree-phase communication, specifically solids, liquids and gases,between freezing section 20 and extraction section 22.

[0094] Optionally, conduit 210 includes three-phase mixing means 216 formixing the three phases in the three-phase flow, whereby at least aportion of frozen microdroplets 44 contained in the gaseous phase willbe captured in the liquid phase, thereby increasing product yield byreducing the loss of frozen microdroplets 44 from venting gases throughgas outlet 52. Suitable three-phase mixing means 216 include a cascadingbaffle, or preferably, one or more elements of a static mixer (e.g.,Model # KMR-SAN; Chemineer, Inc.). A preferred three-phase mixing means216 provides a tortuous flow. More preferably, three-phase mixing means216 comprises a number of in-series static mixer elements sufficient tocreate turbulent flow, typically four elements.

[0095] In a further embodiment, solution source 40, includes mix tank218, having a second mixing means (not shown) and fragmentation loop222. Any means for mixing a solution, suspension or emulsion is suitablefor a second mixing means. High shear mixing is preferred for the secondmixing means.

[0096] Fragmentation loop 222 includes fragmentation inlet 224, which isdisposed at, or near, the bottom of dispersion tank 218, fragmentationoutlet 226, which is disposed at dispersion tank 218 generally elevatedabove fragmentation inlet 224. Fragmentation loop 222 also includesfragmentation means 228, which is disposed between fragmentation inlet224 and fragmentation outlet 226, and which reduces, or micronizes thesize of particles suspended in the material solution; and which formsfiner, better blended, emulsions of immiscible liquids. Suitablefragmentation means 228 include means capable of fragmenting a solid toa diameter between about 1 micrometer, or less, and about 10micrometers. Examples of suitable fragmentation means 228 includerotor/stator homogenizers, colloid mills, ball mills, sand mills, mediamills, high pressure homogenizers.

[0097] In an alternate embodiment, fragmentation occurs within mix tank218 by the use of disruptive energy, such as that provided by a sonicprobe, high shear mixer or homogenizer.

[0098] The temperature of dispersion tank 218 and/or of fragmentationloop 222 is typically controlled when containing proteins, or other heatsensitive materials, by means known in the art, to minimize proteindenaturing.

[0099] In a method illustrated in FIG. 3, the volatilized gas, liquifiedgas and frozen microdroplets 44 are directed from freezing section 20and through conduit 210, which includes three-phase mixing means 216,preferably a four-element, or more, static mixer, to turbulently mix thethree phases and scrub frozen microdroplets 44, which were entrained inthe gas phase, into the liquified gas, thereby improving yield.

[0100] In another embodiment, a solution containing an additionalsubstance, which is in solid form or which forms an emulsion with thesolvent, is recirculated through fragmentation means 228, such as ahomogenizer, to micronize the solid particles, preferably particulatesof about 1-10 micrometers in diameter, or to further blend the emulsionto form smaller emulsion droplets.

[0101] Fragmentation is not required when the solution has no suspendedparticles, or when larger suspended particles are desired.

[0102] Alternately, the second mixing means can be used as afragmentation means, such as when a high speed/high shear mixer is usedfor the second mixing means.

[0103]FIG. 4 illustrates yet another embodiment of an apparatus of theinvention, suitable for performing the method of invention. Theapparatus of FIG. 4 has many of the same elements of FIGS. 1 and 3, andlike elements are designated with like numerals. This apparatus includesmultiple freezing vessels 202, each containing a separate freezingsection 20. The apparatus also includes one extraction vessel 206,having extraction section 22. Three-phase communication is provided fromeach freezing section 20 to extraction section 22 by separate conduits210. Each conduit 210 includes separate three-phase mixing means 216.

[0104] In the method illustrated in FIG. 4, frozen microdroplets 44 areformed in each freezing section and then transferred to a commonextraction section 22.

[0105] The composition made according to the method of this inventioncan be administered to a human, or other animal, orally, by suppository,by injection or implantation subcutaneously, intramuscularly,intraperitoneally, intracranially, and intradermally, by administrationto mucosal membranes, such as intranasally or by means of a suppository,or by in situ delivery (e.g. by enema or aerosol spray) to provide thedesired dosage of a biologically active agent based on the knownparameters for treatment of various medical conditions.

[0106] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

What is claimed is:
 1. A method for forming microparticles comprisingthe steps of: a) directing microdroplets of a mixture comprising abiocompatible polymer, a solvent for the polymer and a protein, peptideor small molecule, into a freezing section containing a liquefied gas,whereby the microdroplets freeze; and b) contacting the frozenmicrodroplets in an extraction section with a liquid non-solvent toextract the solvent into the non-solvent thereby forming saidmicroparticles; wherein the freezing section and extraction section areseparated, and the non-solvent is in the liquid state throughout themethod.
 2. The method of claim 1, wherein the biocompatible polymer isbiodegradable.
 3. The method of claim 2, wherein said biocompatible andbiodegradable polymer is selected from the group consisting ofpoly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s,poly(lactic acid)s, poly(glycolic acid)s, polycarbonates,polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters,polycaprolactone, poly(dioxanone)s, poly(alkylene alkylate)s,polyurethanes, blends and copolymers thereof.
 4. The method of claim 3,wherein the polymer is a poly(lactide-co-glycolide).
 5. The method ofclaim 1, wherein the temperature of step (b) is lower than thetemperature of step (c).
 6. The method of claim 1, wherein the liquefiedgas is sprayed into the freezing section.
 7. The method of claim 1,wherein the frozen microdroplets are collected at the bottom of thefreezing section and directed into the extraction section.
 8. A methodfor forming microparticles comprising the steps of: a) directing themicrodroplets of a mixture comprising a biocompatible polymer, a solventfor the polymer and a protein, peptide or small molecule, into afreezing vessel containing a liquefied gas, whereby the microdropletsfreeze; and b) contacting the frozen microdroplets in an extractionvessel with a liquid non-solvent to extract the solvent into thenon-solvent thereby forming said microparticles; wherein the freezingvessel and extraction vessel are separated, and the non-solvent is inthe liquid state throughout the method.
 9. The method of claim 8,wherein the biocompatible polymer is biodegradable.
 10. The method ofclaim 9, wherein said biocompatible and biodegradable polymer isselected from the group consisting of poly(lactide)s, poly(glycolide)s,poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,polycarbonates, polyesteramides, polyanhydrides, poly(amino acids),polyorthoesters, polyacetals, polycyanoacrylates, polyetheresters,polycaprolactone, poly(dioxanone)s, poly(alkylene alkylate)s,polyurethanes, blends and copolymers thereof.
 11. The method of claim 8,wherein the polymer is a poly(lactide-co-glycolide).
 12. The method ofclaim 8, wherein the temperature of step (b) is lower than thetemperature of step (c).
 13. The method of claim 8, wherein theliquefied gas is sprayed into the freezing vessel.
 14. The method ofclaim 8, wherein the frozen microdroplets are collected at the bottom ofthe freezing vessel and directed into the extraction vessel.